The invention relates to retroviral vectors, and their use in gene transfer.
The human immunodeficiency virus (HIV) is the etiological agent of the acquired immunodeficiency syndrome (AIDS) and related disorders. The expression of the virus in infected persons is regulated to enable the virus to evade the host""s immune response. The HIV viruses (e.g. HIV-1 and HIV-2), as well as the simian immunodeficiency virus (SIV), share many structural and regulatory genes such as gag, pol, env, tat, rev and nef. See Guyader et al., Nature 328:662-669, 1987, which is incorporated by reference. HIV has been classified as a lentivirus because it causes slow infection, and has structural properties in common with such viruses (Haase. Nature 322:130-136, 1986).
All known retroviruses share features of the replicative cycle, including packaging of viral RNA into virions, entry into target cells, reverse transcription of viral RNA to form the DNA provirus, and stable integration of the provirus into the target cell genome. Replication competent proviruses contain, at a minimum, regulatory long terminal repeats (LTRs) and the gag, pro, pol and env genes which encode core proteins, a protease, reverse transcriptase/RNAse H/integrase and envelope glycoproteins, respectively.
HIV shares the gag, pro, pol and env genes with other retroviruses. HIV-1 also possesses additional genes modulating viral replication, such as the vif, vpr, tat, rev, vpu and nef genes. HIV-2 contains a vpx gene which is not present in HIV-1, but lacks the HIV-1 vpu gene. Additionally the long terminal repeats (LTR) of both HIV-1 and HIV-2 contain cis-acting sequences that are important for integration, transcription and polyadenylation.
HIV, like other retroviruses, are RNA viruses that replicate through a DNA proviral intermediate which is integrated into the genome of the infected host cell. The virion particle contains a dimer of positive strand genomic RNA molecules, which is transcribed from the proviral DNA by the host RNA polymerase II. A portion of these full length RNAs which encode the gag and pol genes of the virus are translated by the host cell ribosomes to produce the structural and enzymatic proteins required for production of virion particles. The provirus also gives rise to a variety of smaller singly and multiply spliced mRNAs coding for the envelope proteins and regulatory proteins.
Wild type retroviruses have been modified to become vehicles for the delivery, stable integration, and expression of cloned genes into a wide variety of cells for experimental and therapeutic purposes. To achieve the aims of transfer and expression of nonviral genes, the vector behaves as a retroviral genome and passes as a virus from a producer cell line. Hence its DNA contains the regions of the wild-type retroviral genome required in cis for incorporation into a retroviral particle. In addition the vector also contains regulatory signals that lead to the optimization of the expression of the cloned gene once the vector is integrated in the target cell as a provirus.
All viral structural genes can be discarded and replaced by heterologous coding sequences, but certain essential sequence elements are retained within the vector. These sequence elements include the packaging sequence, a tRNA binding site, sequences in the LTR that permit xe2x80x9cjumpingxe2x80x9d of the reverse transcriptase between RNA strands during DNA synthesis, sequences near the ends of the LTRs that are necessary for the integration of the vector DNA into the host cell chromosome, and sequences adjoining the 3xe2x80x2 LTR that serve as the priming site for synthesis of the plus strand DNA molecules. See Rapley and Walker, Molecular Biomethods Handbook, 1998, chapter 18 for a discussion of principles of retroviral vector construction, and Lewin, Genes V, 1995, chapter 35, for a discussion of the function of retroviral genes. Since vector genomes do not require that the viral structural genes gag, pol and env be retained, nonviral genes can be cloned into the space vacated by their removal.
A significant advance in the use of retroviral vectors has been the use of packaging cells that stably or constitutively express the viral gag, pol and env genes (for example from plasmids) that cannot themselves be packaged by their own encoded proteins, because they lack the essential packaging sequences. However, when a retroviral transfer vector genome is transfected into such a packaging cell, the viral proteins recognize and package the vector RNA genome into viral particles that are released into the culture supernatant. In such a vector system, the transfer vector (which includes the packaging sequence) shuttles the transgene with the potential for regulation and high titer encapsidation, while the packaging cell line encapsidates the transfer vector RNA but not the viral RNA, so that the packaging cell line does not act as a helper virus. The viral particles produced in this manner can be used to deliver the encapsidated retroviral vector to a target cell with high efficiency.
For HIV-2, it has previously been reported that the leader sequence of this lentivirus contains a packaging signal downstream of the splice donor site (Garzino-Demo et al., Hum. Gene Ther. 6:177-184, 1995). Another report suggested that the downstream sequence elements made only a minor or no contribution to RNA encapsidation, and that the major element was located upstream of the splice donor site (McCann and Lever, J. Virol. 71:4133-4137, 1997). Since a knowledge of the packaging signals of HIV-2 is important to the optimal construction of packaging deficient vectors, this uncertainty about the location of the packaging signals has impeded the use of HIV-2 retroviral vector systems.
Moreover, it would be advantageous to express a transgene using an HIV-2 retroviral vector, in such a manner that packaging of the vector RNA is maximized, without an increase in the packaging of viral RNA.
The invention derives from the discoveries that:
1) deletion of sequences both upstream and downstream of the 5xe2x80x2 splice donor (SD) region of the HIV-2 provirus (packaging vector) results in suppressed encapsidation of packaging vector genomes without critical loss of gene expression, thus the production of xe2x80x9chelper virusxe2x80x9d is suppressed while making adequate structural viral protein available for encapsidation of foreign nucleotides; and
2) that functional deletion of the SD site of the HIV-2 provirus (transfer vector) results in enhanced encapsidation of the transfer vector""s own genome, especially when the host cell has been co-transfected with the packaging vector as described under (1) above; and
3) that the HIV-2, but not HIV-1, packaging vector specifically and faithfully packages its own optimally constructed transfer vector as described under (2) above; and
4) that HIV-2 packaging vector gives both better quality and titer of vector.
Transfer and packaging vectors incorporating one or a combination of these features are useful as gene delivery agents, for example gene therapeutic agents, and provide an improved HIV-2 viral vector system that allows transfer of a transgene into the genome of non-dividing cells. The vectors of the invention also may be used to create a high-efficiency packaging cell line that provides greatly enhanced packaging of foreign DNA, especially when such DNA is carried within the SD deleted transfer vector of the invention. Additionally, it has been discovered that, for the transfer vector of the invention, deletion of the 3xe2x80x2 LTR and its replacement with a puromycin-poly(A) cassette results in still further suppression of encapsidation of packaging virus genomes, without substantial loss of viral particle expression.
The invention includes the transfer vector derived from an HIV lentivirus, such as HIV-2/ST, wherein the vector is functionally deleted for the splice donor site (SD), and contains a functional packaging signal and a transgene operably linked to a promoter. When susceptible cells (such as 293 cells) are co-transfected with the transfer vector and a packaging-defective HIV-2 having a functional deletion of its packaging signal, production of progeny virions is enhanced by deletion of the SD. Alternatively, the transfer vector can be introduced into a packaging cell stably transfected with the packaging vector. In particular embodiments, the lentivirus is HIV-2, the functional deletion of SD comprises nucleotide changes and/or deletions in the SD nucleotide sequence, and the transgene is a neo gene.
In other embodiments, the invention includes a packaging vector derived from HIV-2, such as HIV-2(ROD), comprising a 5xe2x80x2 splice donor site, and an upstream and a downstream packaging signal sequence in the leader sequence, wherein both the upstream and downstream packaging signal sequences are functionally deleted to substantially eliminate packaging of progeny viral RNA, but the splice donor site is functionally intact. In particular examples, the deletions in the packaging sequence comprise no more than 164 nucleotides upstream of the SD and no more than 62 nucleotides downstream of the SD, for example 153 nucleotides (nt 306-458) upstream of the SD, and 52 nucleotides (nt 486-538) downstream of the SD. In particular examples, each deletion is at least 5, 10, 20, 50 or 100 nucleotides in length.
In other examples, the upstream packaging signal is contained in nucleotides downstream from nucleotide 300 and upstream from the SD, and the downstream packaging signal corresponds to nucleotides downstream from the SD and upstream from nucleotide 539. The packaging vector may also include a 3xe2x80x2 LTR that is functionally deleted, for example by replacement of the 3xe2x80x2 LTR with a heterologous transcriptional termination sequence.
In an alternative embodiment, the HIV packaging vector (or stably transfected cell) includes a polynucleotide sequence which encodes HIV proteins (such as HIV-2 proteins), wherein the polynucleotide sequence includes a mutation in a leader sequence upstream from a 5xe2x80x2 splice donor site, and a mutation between the 5xe2x80x2 splice donor site and an initiation codon of a gag gene, which results in HIV RNA (such as HIV-2 RNA) transcribed from the vector being substantially packaging defective. The polynucleotide sequence may include (a) a DNA segment from an HIV-2 genome, wherein the DNA segment comprises the HIV gag, pot, rev and env genes, and the vector lacks the bipartite HIV-2 packaging sequence necessary to package HIV-2 RNA into virions; (b) an intact 5xe2x80x2 splice donor site; and (c) a promoter operably linked to the DNA segment of (a), wherein the vector, when introduced into or expressed in a eukaryotic host cell, expresses HIV-2 Gag, Pol, Rev, and Env proteins, as well as the Tat protein (if the linked promotor is 5xe2x80x2 LTR), to form HIV-2 virions that are not packaged.
In some embodiments, the transfer vector includes a polynucleotide sequence which encodes a transgene, and an HIV (such as an HIV-2) packaging signal and promoter, but which does not encode one or more of a complete gag, pot, or env gene, and in which the splice donor site is mutated to render it non-functional, which increases encapsidation of the transgene vector RNA, compared to encapsidation of the transgene RNA in the absence of the mutation in the splice donor site. The splice donor site may be mutated to functionally delete it by substantially deleting the site, changing its nucleotides, or deleting a sufficient portion of it to increase encapsidation of the transgene RNA.
The invention also includes a cell that expresses or has been transfected with the transfer vector and/or the packaging vector, or which stably expresses the genome of the packaging vector. In particular examples, the cell is a 293T or SupT cell, the transfer vector is pSGT-5(SDM) and the packaging vector is pROD(SD36). When the cell is transfected with or stably expresses both the transfer and packaging vectors, transgene RNA encapsidation is substantially increased in the presence of the transfer vector with the mutated splice donor site, as compared to transgene encapsidation in the presence of the transfer vector in which the splice donor site is not mutated. In particular examples, the packaging vector is an HIV-2(ROD) clone, such as pROD(SD36) or a combination of envelope defective pROD(SD36/EM) and envelope expression plasmid pCON-ENV(ROD). In addition to parental HIV-2(ROD), HIV-2 envelope is derived from mutant HIV-2 and it can fuse with a broad variety of cells whether they contain CD4 markers or not.
Other embodiments include dividing the packaging vector functionally and structurally into two. The first vector contains all of the necessary elements of a packaging vector, except that its envelope is defective. In particular embodiments, this vector is pROD(SD36/EM) or pCM-ROD(SD36/EM). The second vector provides the envelope in trans, to complement the defect. In particular embodiments, this vector is pCM-VSV-G or pCM-ENV(ROD).
The invention also includes a method for improving encapsidation of transgene RNA using retroviral packaging and transfer vectors by (in any order) introducing into the target cell the transfer vector and packaging vector. Alternatively, the transfer vector can be introduced into a cell that stably expresses an HIV-2 packaging genome that has been rendered packaging deficient by the mutation of both the upstream and downstream packaging signals.
Also included in the invention are functionally equivalent transfer and packaging vectors generated using the SIV genome.
Also included in the invention are the supernatant of the packaging cell that includes the encapsidated vector RNA, and the encapsidated vector RNA itself.